Two B-type cyclins, B1 and B2, have been identified in mammals. Proliferating cells express both cyclins, which bind to and activate p34 cdc2 . To test whether the two B-type cyclins have distinct roles, we generated lines of transgenic mice, one lacking cyclin B1 and the other lacking cyclin B2. Cyclin B1 proved to be an essential gene; no homozygous B1-null pups were born. In contrast, nullizygous B2 mice developed normally and did not display any obvious abnormalities. Both male and female cyclin B2-null mice were fertile, which was unexpected in view of the high levels and distinct patterns of expression of cyclin B2 during spermatogenesis. We show that the expression of cyclin B1 overlaps the expression of cyclin B2 in the mature testis, but not vice versa. Cyclin B1 can be found both on intracellular membranes and free in the cytoplasm, in contrast to cyclin B2, which is membrane-associated. These observations suggest that cyclin B1 may compensate for the loss of cyclin B2 in the mutant mice, and implies that cyclin B1 is capable of targeting the p34 cdc2 kinase to the essential substrates of cyclin B2.Mitotic B-type cyclins activate the p34 cdc2 protein kinase to form maturation promoting factor, which is required for cells to undergo mitosis (1-3). Only two B-type cyclins, B1 (4, 5) and B2 (6, 7), have been identified so far in mammals, although chickens, frogs, flies, and nematode worms possess a third, more distant relative-cyclin B3 (8, 9). The genes of cyclin B1 and B2 show very little similarity in the first 100 residues, and about 57% identity in the remaining 300 residues. The two genes must have diverged early in vertebrate evolution, because frogs have both genes. The family resemblances are strongly conserved: Goldfish B1 is almost 70% identical to mouse B1 in the C-terminal 300 residues, and chicken B2 is 75% identical to mouse B2 in the equivalent region. Fig. 1 shows a dendrogram of the current B-type cyclin family tree, omitting yeast examples that cluster together as a separate branch. Cyclins B1 and B2 are coexpressed in the majority of dividing cells, although their subcellular localization differs, with cyclin B1 usually associated with microtubules, and cyclin B2 with intracellular membranes (10-15). Cyclin B1 enters the nucleus in late G 2 phase of the cell cycle, whereas cyclin B2 does not (15). Cyclins B1 and B2 also show different patterns of expression during murine spermatogenesis (6), and in Xenopus oocytes, cyclin B2 protein is already present in unactivated oocytes, whereas cyclin B1 is not synthesized until progesterone induced oocyte maturation (16,17). These considerations would lead one to suspect that cyclins B1 and B2 have specialized roles in preparing cells for mitosis, but there is no direct evidence for this view from studies in vertebrates.

In vertebrates Cdk1 is required to initiate mitosis; however, any functionality of this kinase during S phase remains unclear. To investigate this, we generated chicken DT40 mutants, in which an analog-sensitive mutant cdk1 as replaces the endogenous Cdk1, allowing us to specifically inactivate Cdk1 using bulky ATP analogs. In cells that also lack Cdk2, we find that Cdk1 activity is essential for DNA replication initiation and centrosome duplication. The presence of a single Cdk2 allele renders S phase progression independent of Cdk1, which suggests a complete overlap of these kinases in S phase control. Moreover, we find that Cdk1 inhibition did not induce re-licensing of replication origins in G2 phase. Conversely, inhibition during mitosis of Cdk1 causes rapid activation of endoreplication, depending on proteolysis of the licensing inhibitor Geminin. This study demonstrates essential functions of Cdk1 in the control of S phase, and exemplifies a chemical genetics approach to target cyclin-dependent kinases in vertebrate cells.

In higher eukaryotes, the S phase and M phase of the cell cycle are triggered by different cyclin-dependent kinases (CDKs). For example, in frog egg extracts, Cdk1–cyclin B catalyzes entry into mitosis but cannot trigger DNA replication. Two hypotheses can explain this observation: Either Cdk1–cyclin B fails to recognize the key substrates of its S-phase–promoting counterparts, or its activity is somehow regulated to prevent it from activating DNA synthesis. Here, we show that Cdk1–cyclin B1 has cryptic S-phase–promoting abilities that can be unmasked by relocating it from the cytoplasm to the nucleus and moderately stimulating its activity. Subcellular localization of vertebrate CDKs and the control of their activity are thus critical factors for determining their specificity.

The anaphase-promoting complex/cyclosome (APC/C) is essential for progression through mitosis. At anaphase onset, the APC/C requires the activator protein CDC20 to target securin and cyclin B1 for proteasome-dependent degradation, but then depends on the CDC20-related protein FZR1 (also known as CDH1) to remain active until the onset of the next S phase. To investigate the role of FZR1 in mammalian cells, we used RNAi in human cell lines and conditional gene targeting in mouse embryonic fibroblasts. In neither case was FZR1 required for exit from mitosis, but in cells lacking FZR1, the G1 phase was shortened and the S phase was prolonged. In several normal and transformed human cell lines, loss of FZR1 function induced DNA-damage responses and impaired proliferation independently of the p53 status. Constitutive knockdown of p53 in U2OS cells with inducible FZR1 siRNA also failed to restore their proliferative capacity. Thus, the proliferation defects are a direct consequence of the genetic damage inflicted by loss of FZR1 function and are largely independent of p53. In summary, mammalian FZR1 is not required for the completion of mitosis, but is an important regulator of G1 phase and is required for efficient DNA replication in human and mouse somatic cells.